15N Stable Isotope Labeling PSTs in Alexandrium minutum for Application of PSTs as Biomarker - MDPI

Page created by Richard Thompson
CONTINUE READING
15N Stable Isotope Labeling PSTs in Alexandrium minutum for Application of PSTs as Biomarker - MDPI
toxins
Article
15 N
   Stable Isotope Labeling PSTs in Alexandrium
minutum for Application of PSTs as Biomarker
Wancui Xie 1,2 , Min Li 1 , Lin Song 1,2 , Rui Zhang 3 , Xiaoqun Hu 1 , Chengzhu Liang 4 and
Xihong Yang 1,2, *
 1    College of Marine Science and Biological Engineering, Qingdao University of Science & Technology,
      Qingdao 266042, China; xiewancui@163.com (W.X.); 2017170007@mails.qust.edu.cn (M.L.);
      lylinsong@hotmail.com (L.S.); 2018170002@mails.qust.edu.cn (X.H.)
 2    Key Laboratory for Biochemical Engineering of Shandong Province, Qingdao 266042, China
 3    College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China;
      zhangr1168@163.com
 4    Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao 266000, China; liangcz@163.com
 *    Correspondence: 03137@qust.edu.cn
                                                                                                     
 Received: 27 February 2019; Accepted: 4 April 2019; Published: 8 April 2019                         

 Abstract: The dinoflagellate Alexandrium minutum (A. minutum) which can produce paralytic shellfish
 toxins (PSTs) is often used as a model to study the migration, biotransformation, accumulation, and
 removal of PSTs. However, the mechanism is still unclear. To provide a new tool for related studies,
 we tried to label PSTs metabolically with 15 N stable isotope to obtain 15 N-PSTs instead of original 14 N,
 which could be treated as biomarker on PSTs metabolism. We then cultured the A. minutum AGY-H46
 which produces toxins GTX1-4 in f/2 medium of different 15 N/P concentrations. The 15 N-PSTs’ toxicity
 and toxin profile were detected. Meanwhile, the 15 N labeling abundance and 15 N atom number of
 15 N-PSTs were identified. The 14 N of PSTs produced by A. minutum can be successfully replaced by
 15 N, and the f/2 medium of standard 15 N/P concentration was the best choice in terms of the species’

 growth, PST profile, 15 N labeling result and experiment cost. After many (>15) generations, the
 15 N abundance in PSTs extract reached 82.36%, and the 15 N atom number introduced into GTX1-4

 might be 4–6. This paper innovatively provided the initial evidence that 15 N isotope application
 of labeling PSTs in A. minutum is feasible. The 15 N-PSTs as biomarker can be applied and provide
 further information on PSTs metabolism.

 Keywords: Alexandrium minutum; dinoflagellate; paralytic shellfish toxins (PSTs); 15 N stable isotope
 labeling; biomarker

 Key Contribution: The 14 N of paralytic shellfish toxins produced by experimental Alexandrium
 minutum can be replaced by 15 N successfully. 15 N abundance in paralytic shellfish toxins extract
 reached 82.36%, and 15 N atom number introduced into Gonyautoxin1-4 may be 4–6. 15 N-paralytic
 shellfish toxins as biomarker have potential application in some fields such as metabolomics and
 clinical mechanism of paralytic shellfish toxins.

1. Introduction
     Harmful algal blooms (HABs) occur frequently in coastal areas worldwide, causing public
concerns. Firstly, HABs cause millions of dollars economic losses in the tourism and industry sectors [1].
Secondly, HABs break the balance of marine ecosystems as they can disrupt communities and food web
structures [2,3]. Thirdly, phycotoxins produced by HABs may be transferred through the food cycle,
thereby causing lethal and sublethal effects on humans [4,5]. Among all the toxins produced by HABs

Toxins 2019, 11, 211; doi:10.3390/toxins11040211                                  www.mdpi.com/journal/toxins
15N Stable Isotope Labeling PSTs in Alexandrium minutum for Application of PSTs as Biomarker - MDPI
Toxins 2019, 11, 211                                                                                 2 of 13

species, paralytic shellfish toxins (PSTs) produced by dinoflagellates are the most widespread and potent
shellfish contaminating biotoxins [6]. They are accumulated by filter-feeding bivalve mollusks and some
zoophagous mollusks [7]. One of the most widespread toxigenic microalgal taxa is the dinoflagellate
Alexandrium, and studies have demonstrated that toxic Alexandrium spp. disrupt behavioral and
physiological processes in marine filter feeders [8,9]. In recent years, A. tamarense, A. minutum,
A. ostenfeldii, and others were used as carriers of PSTs to track the migration, biotransformation,
accumulation, and removal of PSTs under experimental conditions [10–12]. However, the metabolism
of PSTs in bivalves is still unclear. Thus, there is a need for a new powerful tool to conduct
PST-related researches.
     Isotopic tracer technique can be used to study the absorption, distribution, metabolism, and
excretion of some substances, such as protein and lipid, or to establish organism trophic links in
the ecosystem [13–16]. The stable isotope labeling (ICAT, ICPL, IDBEST, iTRAQ, TMT, IPTL, and
SILAC)-based quantitative approaches are highly efficient for obtaining highly accurate quantification
results and for building more extensive small molecule databases [14,17], especially in proteomics [18,19].
Some studies were conducted on microalgae due to their ability to fix stable isotopes photosynthetically
into cells and offer various target products [20–23]. Artificially enriched stable isotopes of nitrogen can
be fed to cells, either in the form of 15 N-labeled amino acid or 15 N-labeled inorganic salt. According
to [24], the microalgae Chlamydomonas reinhardtii was used to produce 15 N-labeled amino acids with a
high isotopic enrichment and sixteen 15 N-labelled amino acids could be obtained.
     However, isotope-labeling toxin-producing algae have been rarely studied so far. In the present
study, we metabolically labeled the A. minutum produced gonyautoxins1-4 (GTX1-4) that belonging
to mono-sulfated subgroup of PSTs [25]. The culture was carried out with 15 N-NaNO3 as nitrogen
source to obtain the 15 N-PSTs that could be used as biomarker and provide further information on
PSTs metabolism in filter feeding bivalve.

2. Results

2.1. Batch Culture:    15 N/P   Influence in Algal Growth and Toxicity
      Initial batch culture experiments allowed comparison of algae growth under different 15 N/P
conditions, as follows: Group A: 1.0 time of the f/2 medium standard 15 N/P concentration; Group B: 1.5
times of the f/2 medium standard 15 N/P concentration; Group C: 2.0 times of the f/2 medium standard
15 N/P concentration; Group D: 2.5 times of the f/2 medium standard 15 N/P concentration; and Group E:

3.0 times of the f/2 medium standard 15 N/P concentration. The results showed that 15 N/P concentration
can affect algal growth (Figure 1a). In an optimal culture environment, the growth curve of algae cell
can be roughly divided into 4 phases, as follows: lag phase 0–15 days; log phase 16–30 days; 31 days of
the stable phase; and decay phase. In this study, the decay phase was not studied, because the focus
was on the degree of 15 N labeling of algal cellular toxin. The results were similar to those obtained in
the report [26]. However, the lag phase of this experiment was much longer, and it may have been
caused by partial mechanical damage to cells when algal cells were collected by centrifugation. After
the growth of algal cells into the log phase, Group B cell density was higher than those of the other
four groups (p < 0.05). At 30 days, the maximum value of Group B reached 3.820 × 104 cells/mL, and
there was no significant difference in Group A cell density (3.342 × 104 cells/mL) (p > 0.05). However,
the biomass of Groups A and B biomass was much higher than that of the other three groups (p < 0.01).
Lower biomass at high nutrient concentration could be attributed to the inhabitation of photosystem
II’s photosynthetic capacity at high nutrient levels [27].
      15 N/P conditions also can affect toxicity of algae cells (Figure 1b). The highest toxin levels were

determined at day 25 in the log phase in all groups, the same as [28], not early- or post- stationary
growth phase mentioned in [29,30]. Concerning the factors influencing toxicity, the toxin content per
cell in batch culture was not only related to the cell growth stage, but was also affected by intracellular
Toxins 2019, 11, 211                                                                                                            3 of 13

 Toxins 2019, 11, 211                                                                                                          3 of 12
  nutrient salts (e.g., nitrogen, phosphorus, and carbon dioxide), thereby reflecting the balance between
Toxins 2019, 11,and
  synthesis      211 leakage of toxins (e.g., catabolism and cell division) [31].                  3 of 12

                                     (a)                                                              (b)
                                  (a)                                                                (b)
       Figure 1. The effects of 15N/P concentration on cell growth (a) and toxicity (b) in batch culture. (——,
                                             15 N/P
       −−−−,
        Figure
      Figure      TheThe
              ∙∙∙∙∙∙∙∙,
              1. 1.     −∙−∙−∙effects
                         effects and     of
                                    of 15−∙∙−∙∙−∙∙:  concentration
                                                               on cellongrowth
                                                    Growth/Sigmoidal.
                                          N/P concentration               cell growth
                                                                          Letters    and(a)
                                                                                 (a)indicate and  toxicity
                                                                                              significant
                                                                                         toxicity           (b) in batchbetween
                                                                                                           differences
                                                                                                   (b) in batch  culture. culture.
                                                                                                                          (——,
        (——,    −−−−,
       conditions).        ········, −·−·−·    and  −··−··−··: Growth/Sigmoidal.       Letters  indicate  significant
      −−−−, ∙∙∙∙∙∙∙∙, −∙−∙−∙ and −∙∙−∙∙−∙∙: Growth/Sigmoidal. Letters indicate significant differences between        differences
        between conditions).
      conditions).
 2.2. Generation to Generation Culture: 1515       N/P Influence in Algal Growth and Toxicity
  2.2. Generation to Generation Culture:15 N/P Influence in Algal Growth and Toxicity
2.2. Generation
       The algaltocells
                     Generation     Culture:
                            of Groups      A and N/PBInfluence    in Algal
                                                        were chosen        viaGrowth    and Toxicity
                                                                                 generation      to generation culture (three
        The algal cells of Groups A and B were chosen                      via   generation       to generation culture (three
 generations)
      The algalbecause
                   cells ofofGroups
                               better growth.
                                          A and BThe     effects
                                                       were        of 15N/P
                                                              chosen  15
                                                                         via concentration
                                                                               generation to on        cell growth
                                                                                                   generation        and toxicity
                                                                                                                 culture   (three
  generations) because of better growth. The effects of15 N/P concentration on cell growth and toxicity
 are  displayed   in  Figure    2.  In the   first generation,    Group     B   algae  cell  growth
generations) because of better growth. The effects of N/P concentration on cell growth and toxicity     was   better than   that of
  are displayed in Figure 2. In the first generation, Group B algae cell growth was better than that
 Group     A; the  same     results   were     obtained    in batch    culture     experiments.
are displayed in Figure 2. In the first generation, Group B algae cell growth was better than that    In  the second    and   third
                                                                                                                                of
  of Group A; the same results were obtained in batch culture experiments. In the second and third
 generations,
Group     A; the the
                  same cellresults
                             density    of Group
                                     were    obtained  B was    less culture
                                                         in batch    than that       of Group A,
                                                                                  experiments.       In especially
                                                                                                        the secondinand  thethird
                                                                                                                              third
  generations, the cell density of Group B was less than that of Group A, especially in the third generation.
 generation. Interestingly,
generations,    the cell density   there
                                       of was
                                           Group no significant
                                                     B was lessdifference
                                                                    than that (pof< Group
                                                                                        0.05) inA,  algal  cytotoxicity
                                                                                                       especially  in the between
                                                                                                                             third
  Interestingly, there was no significant difference (p < 0.05) in algal cytotoxicity between the two groups
 the two groups
generation.          in all three
              Interestingly,         generations.
                                 there                The experimental
                                         was no significant       difference   results  showed
                                                                                 (p < 0.05)          the algae
                                                                                               in algal         cells cultured
                                                                                                          cytotoxicity  betweenin
  in all three generations. The experimental            results showed the algae cells          cultured in a nutrient solution
 a nutrient
the           solution
     two groups     in all with
                            threeagenerations.
                                     higher 15N/P      than
                                                     The      f/2  medium
                                                          experimental          standard
                                                                             results   showed15N/P
                                                                                                   theconcentration    gradually
                                                                                                        algae cells cultured    in
  with a higher 15 N/P than f/2 medium             standard   15 N/P concentration        gradually      deteriorated, indicating
 deteriorated,    indicating
a nutrient solution with a higher that   f/2   medium
                                              15           standard      15N/P concentration           was
                                                N/P than f/2 medium standard N/P concentration gradually
                                                                                            15                more   suitable   for
  that f/2 medium standard 15 N/P            concentration was more           suitable   for  domesticating      high  abundance
 domesticating
deteriorated,      high abundance
                 indicating
  15 N-PST A. minutum.          that f/2 N-PST
                                          15
                                              medium  A. minutum.
                                                          standard 15N/P concentration was more suitable for
domesticating high abundance N-PST A. minutum.
                                        15

                                 (a)                                                                (b)

        Figure 2.2. Effects
                               (a)
                     Effectsofof1515 N/Pconcentration
                                         concentrationononcell
                                                           cellgrowth
                                                                growth(a)
                                                                       (a)and
                                                                           andcell
                                                                               celltoxicity
                                                                                    toxicity(b)
                                                                                             (b)in
                                                                                                  (b)
                                                                                                 in generation
                                                                                                     generation to
                                                                                                                 to
       Figure                     N/P
        generationculture.
       generation     culture.
      Figure  2. Effects of 15N/P concentration on cell growth (a) and cell toxicity (b) in generation to
       15 N/P Effect
  2.3.generation    culture.
                       on Algae PSTs Profile
 2.3. 15N/P Effect on Algae PSTs Profile
        The experiment
2.3. 15N/P                 was   carried   out using 1.0 and 1.5 times of the f/2 medium standard 15 N/P
       The Effect on Algaewas
            experiment      PSTs  Profile out
                                carried         using 1.0 and 1.5 times of the f/2 medium     15 N/Pstandard 15N/P
  concentration compared with 1.0 and 1.5 times of the f/2 medium standard                             concentration.
 concentration    compared    with  1.0 and   1.5 times
                                                      1.0of the 1.5
                                                                f/2 medium  of standard  15N/P concentration.   The
  TheThe   experiment  A.was   carried  outproduced
                                              using       and        times      the f/2 mediumare standard
                                                                                                             15N/P
        experimental       minutum    only               GTX1-4;    the major    components        GTX-2,-3, which
 experimental
concentration     A.  minutum
                compared     withonly   produced     GTX1-4;    the   major   components     are  GTX-2,-3,  which
                                of1.0
                                    theand   1.5toxin
                                                 times(Figure
                                                        of the f/2
                                                                3).medium     standard    N/P concentration.   The
                                                                                        15
  accounted for    about 74%            total                        Changing     the standard   N/P concentration
 accounted forA.about
experimental              74% of
                     minutum       theproduced
                                only    total toxinGTX1-4;
                                                      (Figure the
                                                                3). Changing
                                                                     major       the standard
                                                                             components     are N/P   concentration
                                                                                                 GTX-2,-3,  which
  influenced the profile of PSTs, and no significant difference was found between 1.0 time of the f/2
 influencedfor
accounted     theabout
                   profile of PSTs,
                         74%   of theand
                                       totalnotoxin
                                                significant
                                                     (Figuredifference   was found
                                                               3). Changing            betweenN/P
                                                                                the standard     1.0 concentration
                                                                                                     time of the f/2
 medium     standard   15N/P and 14N/P concentration, indicating that the application of 15N isotope
influenced the profile of PSTs, and no significant difference was found between 1.0 time of the f/2
 labeling can
medium         feasibly
           standard      be used
                      15N/P   and to  studyconcentration,
                                   14N/P      PSTs production     and metabolism.
                                                              indicating   that the application of 15N isotope
labeling can feasibly be used to study PSTs production and metabolism.
Toxins 2019, 11, 211                                                                                                   4 of 13

medium standard 15 N/P and 14 N/P concentration, indicating that the application of 15 N isotope labeling
Toxins 2019, 11, 211                                                                                4 of 12
can feasibly be used to study PSTs production and metabolism.

                       Figure 3. The effects
                       Figure 3.             of 15
                                     effects of 15N/P concentration on A. minutum PST profile.

2.4. 15 N Labeling Abundance Change of 15 N-PSTs
2.4. 15N Labeling Abundance Change of 15N-PSTs
      The 15 N labeling abundance was calculated according to the following formula:
      The 15N labeling abundance was calculated according to the following formula:
                            15 N atom%  = 1/(2I28 /I29 + 1) × 100 (15 N atom%    < 10%);
                                     15N atom%
                             15 N atom% = 2/(I /I= 1/(2I28/I29+1) ×15100 (15N atom% 10%)                                          (1)
                                           15N atom% = 2/(I29/I30+2) × 100 (15N atom%>10%)
      In the algal culture environment, 15 N-NaNO3 of f/2 medium is not the only nitrogen source
because     of the  inorganic      14 N existing in15natural seawater. A. minutum cells prefer absorbing light
       In the    algal  culture environment,            N-NaNO3 of f/2 medium is not the only nitrogen source
elements      14
             ( the
                 N) and    rejecting14Nheavy   elements     (15 N), resultingA.in minutum
                                                                                  nitrogen stable   isotope  fractionation.
because of           inorganic           existing   in natural    seawater.                  cells prefer  absorbing    light
With    increasing     cell density    and   size,  less  and   less 14 N can be utilized. Thus, more 15 N can enter
elements ( N) and rejecting heavy elements ( N), resulting in nitrogen stable isotope fractionation.
              14                                             15

cells,
With and     nitrogen
        increasing        stable
                       cell       isotopes
                            density          fractionation
                                       and size,  less and lessweakens.
                                                                   14N canTwo   culture methods
                                                                            be utilized. Thus, more and15N
                                                                                                         gascan
                                                                                                              isotope
                                                                                                                 enter mass
                                                                                                                        cells,
spectrometer       were   used   to   determine    the  relationship    between   15 N-labeling abundance and culture
and nitrogen stable isotopes fractionation weakens. Two culture methods and gas isotope mass
time,   as shownwere
spectrometer         in Table
                           used1.toDuring     the batch
                                      determine            culture, 15 Nbetween
                                                    the relationship      abundance    was positively
                                                                                   15N-labeling          related
                                                                                                  abundance       to culture
                                                                                                                and   culture
time
time,and     reached
         as shown     in the highest
                         Table          valuethe
                                 1. During      at batch
                                                   day 30.    There 15are
                                                           culture,       significant differences
                                                                       N abundance                  (p 90%).
                                                                 below the abundance of the labeling material 15N-
NaNO3 (δ15N = 99.14%), not reaching our expectation (>90%).
      Table 1. 15 N labeling abundance change of PSTs along with the change of culture time in different
      cultivation
      Table 1. 15Nmethods.
                    labeling abundance change of PSTs along with the change of culture time in different
      cultivation methods.                                          1.0 Time of the f/2           1.5 Times of the f/2
                                                                  Medium Standard           Medium Standard
                       Culture Method                       1.0 Time
                                                                 15 N/Pof the f/2    1.5 Times
                                                                                           15 N/Pof the f/2
                                                                        Concentration             Concentration
                                                            Medium Standard           Medium Standard
                       Culture Method                      15N/P Concentration
                                                                                15
                                                                           PSTs N Abundance    (Atom%)
                                                                                    15N/P Concentration

                                    0                             PSTs 150.47
                                                                           N Abundance (Atom%) 0.47
       Batch culture/d             20                                     26.26                      26.43
                                                    0               0.47                     0.47
                                   30                                     57.16                      70.26
                Batch culture/d     1              20               26.26 37.60             26.43 -
 Generation to generation
                                    2              30               57.16 58.32             70.26 -
   culture/generation
                                    3               1               37.60 62.46                -       -
          Generation to generation -
    No. n generation                                                      82.36
                                                    2               58.32                      -
                  culture/generation
                                                    3               62.46                      -
                   No. n generation                 -               82.36

2.5. The Efficiency of 15N-PSTs Separation and Purification
       N-PSTs extracts were separated and purified by column chromatography on the Bio-Gel P-2
      15

and the weak cation exchanger Bio-Rex 70. In the process of column chromatography on the Bio-Gel
P-2, fluorescence detection and UV absorbance detection were carried out (Figure 4a). The UV
Toxins 2019, 11, 211                                                                                                5 of 13

 2.5. The Efficiency of 15 N-PSTs Separation and Purification
       15 N-PSTs extracts were separated and purified by column chromatography on the Bio-Gel P-2 and
Toxins 2019, 11, 211                                                                                  5 of 12
 the weak cation exchanger Bio-Rex 70. In the process of column chromatography on the Bio-Gel            P-2,
 fluorescence detection and UV absorbance detection were carried out (Figure 4a). The UV absorption
absorption peak did not coincide with the fluorescence absorption peak. Thus, the UV absorption
 peak did not coincide with the fluorescence absorption peak. Thus, the UV absorption signal had
signal had no relationship with the toxin component. As shown by the fluorescence absorption peak,
 no relationship with the toxin component.       As shown by the fluorescence absorption peak, Bio-Gel
Bio-Gel P-2 effectively separated
                             15
                                      15N-PSTs with impurities in crude extracts, such as proteins and
 P-2 effectively separated N-PSTs with impurities in crude extracts, such as proteins and pigments.
pigments. The liquid of fluorescence absorption peak was collected, freeze-dried (10 mg), and
 The liquid of fluorescence absorption peak was collected, freeze-dried (10 mg), and redissolved
redissolved with 0.05 M Tri-HCl (2 mL). The redissolved sample (1 mL) was used for purification by
 with 0.05 M Tri-HCl (2 mL). The redissolved sample (1 mL) was used for purification by column
column chromatography on weak cation exchanger Bio-Rex 70 at gradient elution condition and was
 chromatography on weak cation exchanger Bio-Rex 70 at gradient elution condition and was separated
separated   (Figure
 (Figure 4b).  After 4b). Afterthe
                     analysis,  analysis,
                                   Peak I the
                                           wasPeak Ⅰ was determined
                                               determined              to be the
                                                           to be the isomer      isomer
                                                                              mixture of mixture
                                                                                         GTX1/4, of
                                                                                                 andGTX1/4,
                                                                                                     Peak II
and  Peak  Ⅱ was  the isomer   mixture
 was the isomer mixture of GTX2/3.      of GTX2/3.

                               (a)                                                      (b)
                  Gonyautoxin1-4purified
       Figure4.4.Gonyautoxin1-4
      Figure                     purifiedby
                                          bycolumn
                                             columnchromatography
                                                    chromatographyon
                                                                   onthe
                                                                      theBio-Gel
                                                                          Bio-GelP-2
                                                                                  P-2(a)
                                                                                      (a)and
                                                                                          andthe
                                                                                              theweak
                                                                                                 weak
       cation exchanger BioRex 70 (b).
      cation exchanger BioRex 70 (b).
 2.6. 15 N Atom Number Identification of 15 N-PSTs
2.6. 15N Atom Number Identification of 15N-PSTs
       As demonstrated previously [32], GTX1-4 (Figure 5) can be ionized in ESI positive ion mode,
      As demonstrated previously [32], GTX1-4 (Figure 5) can be ionized in ESI positive ion mode,
 thereby giving abundant fragment ions (Table 2). When 14 N of PSTs is replaced by 15 N, [M+H]+
thereby giving abundant fragment ions (Table 2). When        14N of PSTs is replaced by 15N, [M+H]+ and
 and fragment ions will change with the number of 15 N atoms, so that primary mass spectrum can
fragment ions will change with   the number of N atoms, so that primary mass spectrum can be used
                                                  15
 be used to determine the 15 N atom number of 15 N-PSTs. After the addition of artificial nitrogen in
to determine the 15N atom
                       15   number of 15N-PSTs. After the addition of artificial nitrogen in A. minutum
 A. minutum culture, N was successfully introduced to PSTs, and characteristic fragment ions were
culture, 15N was successfully introduced      to PSTs, and characteristic fragment ions were produced.
 produced. There was no [M+H]+ peak in the mass spectrum as the result of high capillary voltage
There was no [M+H] peak in the mass spectrum as the result of high capillary voltage in this
                        +
 in this experiment (Figure 6). GTX1 produced three characteristic fragment ions (m/z = 334.2966;
experiment (Figure 6). GTX1 produced three characteristic fragment ions (m/z=334.2966; 318.3007;
 318.3007; 319.3054) with the loss of -SO3 or -SO3 -H2 O. GTX4 generated three characteristic fragment
319.3054) with the loss of -SO3 or -SO3-H2O. GTX4 generated three characteristic fragment ions
 ions (m/z = 318.3007; 319.3054; 338.3418) with the loss of -SO3 -H2 O or -SO3 and had two fragment
(m/z=318.3007; 319.3054; 338.3418) with the loss of -SO3-H2O or -SO3 and had two fragment ions
 ions (m/z = 318.3007; 319.3054) similar to those of GTX1. There were four characteristic fragment ions
(m/z=318.3007; 319.3054) similar to those of GTX1. There were four characteristic fragment ions
 (m/z = 322.1197; 321.1252; 305.1583; 317.0497) in GTX2 with the loss of -SO3 or -SO3 -H2 O, whereas
(m/z=322.1197; 321.1252; 305.1583; 317.0497) in GTX2 with the loss of -SO3 or -SO3-H2O, whereas two
 two characteristic fragment ions (m/z = 302.3054; 303.3095) were obtained with the loss of -SO3 -H2 O.
characteristic fragment ions (m/z=302.3054; 303.3095) were obtained with the loss of -SO3-H2O.
 Theoretically, the m/z of fragment ions can increase (+1,+2,+3,+4,+5,+6,+7) corresponding with the
Theoretically, the m/z of fragment ions      can increase (+1,+2,+3,+4,+5,+6,+7) corresponding with the
 number (1,2,3,4,5,6,7) of introduced 15 N atoms in PSTs. Based on these data, 2~6 15 N atoms can be
number (1,2,3,4,5,6,7)15of introduced N atoms in15PSTs. Based on these data, 2~6 15N atoms can be
                                        15
 introduced into the N-PSTs molecule, and 4~6 N atoms is most possible.
introduced into the 15N-PSTs molecule, and 4~6 15N atoms is most possible.

                               Table 2. Mass spectral data for gonyautoxins (GTX1-4).

                   Molecular                                                              Fragment Ion after
       Toxin                         [M+H]+   Fragment Ion            Loss of
                   Formula                                                                    Labeling

       GTX1       C10H17N7O9S         412       332; 314          -SO3; -SO3-H2O              334.3; 318.3; 319.3

                                                                  -SO3; -SO3-H2O;
       GTX4       C10H17N7O9S         412     332; 314;253                                    318.3; 319.3; 338.3
                                                                -SO3-H2O-NH3-CO2

       GTX2       C10H17N7O8S         396       316; 298          -SO3; -SO3-H2O;             322.1; 321.1; 305.1

                                                                 -SO3; -SO3-H2O;
       GTX3       C10H17N7O8S         396     316; 298; 220                                      302.3; 303.3
                                                              -SO3-2H2O-NH3-NHCO
Toxins 2019, 11, 211                                                                                                                                                                               6 of 13

                                           Table 2. Mass spectral data for gonyautoxins (GTX1-4).

                               Molecular                                                                                                                                         Fragment Ion after
     Toxin                                                        [M+H]+                          Fragment Ion                                    Loss of
                               Formula                                                                                                                                               Labeling
     GTX1                   C10 H17 N7 O9 S                                 412                         332; 314                     -SO3 ; -SO3 -H2 O                            334.3; 318.3; 319.3
                                                                                                                                     -SO3 ; -SO3 -H2 O;
     GTX4                   C10 H17 N7 O9 S                                 412                     332; 314;253                                                                  318.3; 319.3; 338.3
                                                                                                                                   -SO3 -H2 O-NH3 -CO2
     GTX2                   C10 H17 N7 O8 S                                 396                         316; 298                     -SO3 ; -SO3 -H2 O;                           322.1; 321.1; 305.1
                                                                                                                                     -SO3 ; -SO3 -H2 O;
     GTX3            C H N O S                                              396                    316; 298; 220                                                                        302.3; 303.3
Toxins 2019, 11, 211 10 17 7 8                                                                                                    -SO3 -2H2 O-NH3 -NHCO                                           6 of 12
Toxins 2019, 11, 211                                                                                                                                                                               6 of 12
                                                              OCONH2                                                       OCONH2
                                                               OCONH2                                                       OCONH2
                                             HO                                   H                       HO                                H
                                                              6                   N                                                         N
                                                                                  7H
                                                                                                                           6
                                              HO     N1                            N
                                                                                                           HO      N1                       7H
                                                                  6                                                         6                N
                                                     N1                            7                               N1                        7
                                                                                                NH2+                                                    NH2+
                                                      2                           9              NH2+                   2                   9            NH2+
                                                              3                                                            3
                                                          2                       N9                                     2                  N9
                                           +H2N               N3                         OH             +H2N               N3                     OH
                                                                                  HN                                                        HN
                                            +H2N               N             12 H  OH                    +H2N              N          12           OH
                                                                      10          OH                                                         H
                                                                              12                                                10     12         OH
                                                                       10                OH                                      10                OH
                                                                             11                                                       11
                                                                         H 11 OSO3-                                       -O3SO 11 H
                                                                          H    OSO3-                                       -O3SO    H
                                                                         GTX1                                               GTX4
                                                                         GTX1                                               GTX4

                                                              OCONH2                                                       OCONH2
                                                               OCONH2                                                       OCONH2
                                                H                                 H                        H                                H
                                                              6                   N                                        6                N
                                                 H   N1                           7H                        H      N1                       7H
                                                               6                   N                                        6                N
                                                     N1                            7                               N1                        7
                                                                                                NH2+                                                    NH2+
                                                      2                           9              NH2+                   2                   9            NH2+
                                                         3                                                                 3
                                                       2                          N9                                     2                  N9
                                          +H2N           N3                              OH             +H2 N              N3                     OH
                                                                                  HN                                                        HN
                                           +H2N               N              12 H  OH                    +H2 N             N          12           OH
                                                                                                                                             H
                                                                   10         12  OH                                            10     12         OH
                                                                    10                   OH                                      10                OH
                                                                             11                                                       11
                                                                       H 11 OSO3-                                         -O3 SO 11 H
                                                                        H    OSO3-                                         -O3 SO    H
                                                                         GTX2                                               GTX3
                                                                         GTX2                                               GTX3
                                          Figure 5. The molecular structural formula of GTX1-4.
                                          Figure 5. The molecular structural formula of GTX1-4.

                                                334.2926                               GTX1                                                                                                   GTX4
    100                                                                                GTX1                     100                                                                           GTX4
                                                 334.2926                                                                                          318.3007
     100                                                                                                         100
                                                                                                                                                    318.3007
     80                                                                                                          80
      80                                                                                                          80
                290.2709
                 290.2709
     60                                                                                                          60
      60                                                                                                          60
    %%

                                                                                                             %%

     40                                                                                                          40
      40                                                                                                          40

     20                       318.3007                                                                           20
      20            291.2723 318.3007                                             362.3250                        20                                             319.3054
                     291.2723          319.3054                                    362.3250                                                                                                338.3418
                                        319.3054                                                                                                                  319.3054                  338.3418
      0                                                                                                            0
       0                                                                                                            0
         280     290    300     310    320   330     340              350         360     370
          280     290    300     310    320
                                                                                                                   300           305        310      315        320       325      330      335    340
                                          m/z 330     340              350         360     370
                                                                                                                    300           305        310      315        320
                                                                                                                                                                m/z        325      330      335    340
                                          m/z                                                                                                                   m/z
                                         (a)                                                                                                                   (b)
                                          (a)                                                                                                                   (b)
                                                                                         GTX2                                                                                                  GTX3
    100                                                                                  GTX2               100                                   302.3054
                                                              322.1197                                                                                                                         GTX3
     100                                                                                                     100                                   302.3054
                                                               322.1197
     80                                   321.1252
                                           321.1252                                                              80
      80                                                                                                          80
     60
                                                                                                                 60
      60
    %%

                                                                                                                  60
                                                                                                            %%

     40     305.1583
      40     305.1583                                                                                            40
                                                                                                                  40
     20            307.1704            317.0497                              328.2077                            20
      20            307.1704            317.0497                              328.2077                            20                              303.3095                                  346.3338
      0                                                                                                                                            303.3095                                  346.3338
       0                                                                                                          0
                                                                                                                   0      290              300          310       320            330       340     350
                305         310        315      320                    325               330
                                                                                                                           290              300          310    m/z 320           330       340     350
                 305         310        315 m/z 320                     325               330
                                            m/z                                                                                                                 m/z

                                         (c)                                                                                                                   (d)
                                          (c)                                                                                                                   (d)
                 Figure6.6. Mass          of 15
                            Mass spectrum of                15N-GTX4
                                              15N-GTX1 (a), 15
                                                               N-GTX4(b),   15
                                                                             N-GTX2
                                                                      (b),1515 N-GTX2(c)   and151515
                                                                                       (c)and     N-GTX3
                                                                                                     N-GTX3(d).
                  Figure 6. Massspectrum
                 Figure           spectrum of 15N-GTX1 (a), 15N-GTX4  (b),    N-GTX2  (c)  and     N-GTX3    (d).
                                                                                                            (d).

3. Discussion
3. Discussion
     Numerous studies have focused on the bioaccumulation and biotransformation of PSTs using
     Numerous studies have focused on the bioaccumulation and biotransformation of PSTs using
Alexandrium strains, such as A. minutum in marine organisms [33]. Stable isotopes have been often
Alexandrium strains, such as A. minutum in marine organisms [33]. Stable isotopes have been often
used to study lipid synthesis, proteomics and ecosystem [13,15,34] and rarely applied to toxin-
Toxins 2019, 11, 211                                                                                7 of 13

3. Discussion
     Numerous studies have focused on the bioaccumulation and biotransformation of PSTs using
Alexandrium strains, such as A. minutum in marine organisms [33]. Stable isotopes have been often used
to study lipid synthesis, proteomics and ecosystem [13,15,34] and rarely applied to toxin-producing
algae [35]. This study intends to use the biosynthetic process to replace the nitrogen atom of A. minutum
with a stable isotope 15 N to form a tracer-enabled A. minutum for PST synthesis and metabolism.

3.1. Effect 15 N/P of on A. minutum Culture
      Growth and toxin production of toxic dinoflagellates vary with nutrients supply. High nutrient
cultures can inhibit the photosynthetic capacity of photosystem II, which related to algae growth [27].
The N:P ratio can be used as an index for the nutritional status and physiological behavior of
phytoplanktons [36]. Kinds of nitrogen sources and N:P supply ratio can affect the physiological
responses of a tropical Pacific strain of A. minutum, the cellular toxin quota (Qt) was higher in
P-depleted, nitrate-grown cultures [37]. To assure the feasibility of isotope 15 N, some experiments
were carried out, including the comparison of different 15 N:P ratio. In batch culture, five 15 N:P ratios
were compared, and the growth of A. minutum under 1.0 and 1.5 times of the f/2 medium standard
15 N/P concentration was better than others (Figure 1a). Similar previous findings reported that cell

densities and growth rates of A. minutum were severely suppressed under high N/P ratios (>100) in
both N-NO3 and N-NH4 treatments [38]. Some studies showed that the incorporation of 15 N-labeled
salt did not affect the growth of green alga Chlamydomonas reinhardtii [24,39], and our study achieved
the same result with A. minutum. The toxin profile of this A. minutum strain is relatively stable and
predominantly constitutes GTX1-4 (Figure 3), the same as four strains of A. minutum collected from
southern Taiwan [40], even under different N:P supply ratios. The highest algae toxicity per cell of all
five groups was observed at day 25, and cellular toxin quota of the exponential growth phase was
higher than that of the stable phase, even though the total number of stable cells is highest during
the entire growth process (Figure 1b). The explanation could be that there was a negative correlation
between algae toxicity per cell and cell density. In other words, the cell size was smaller when cell
density was higher; thus, toxicity per cell of stable phase was lower. As previously reported, changes of
nutrient availability with time in batch culture caused growth stage variability in toxin content, which
peaked during mid-exponential growth [31]. Total toxicity, toxicity per cell, and the number of and
relative proportion of toxin analogs changed in relation to the 15 N:P ratio. The f/2 medium standard
15 N/P concentration at 1.0 time was a better choice to label PSTs with 15 N regardless of cultivation

method, i.e., batch culture or generation to generation culture, for A. minutum growth, PST production
and profile, and experimental cost.

3.2. The Replacement of Stable Isotope 15 N
      The successful production PSTs of labeled substances from A. minutum was detected by MAT-271
Gas isotope mass spectrometer to determine 15 N abundance, Analysis by HPLC-MS was performed
to identify 15 N atom number. In batch culture, δ15 N of two groups’ PSTs had a significant difference
(p < 0.01) in different growth stages (lag, log, and stable phases) (Table 2). Combining 15 N labeling
abundance with mass spectrum results, it can be presumed that artificial 15 N addition can bring 2~6
15 N atoms into the 15 N-PSTs molecule, and the 4~6 15 N atoms’ replacement becomes most possible.

Recently, 15 N stable-isotope-labeling was applied to the toxic dinoflagellate Alexandrium catenella and
the relationship between the order of 15 N incorporation % values of the labeled populations and the
proposed biosynthetic route was established [35]. Relative abundance % of m+6 and m+7 isotopomers
of PSTs were the highest in A. catenella after a two month passage in 15 N-NaNO3 medium in [35],
which is similar to our conclusion. Nitrogen (N) isotopic compositions of PSTs in A. minutum cells
reflect the isotopic fractionations associated with diverse biochemical reactions. Based on PSTs being a
secondary metabolite, a small part of the supplied nitrogen was assimilated into PSTs, and most of
Toxins 2019, 11, 211                                                                                  8 of 13

the nitrogen may participate in the synthesis of other nitrogen compounds. Some findings on high
abundance in biomass (not extracted) have been reported; a process for the cost-effective production of
13 C/15 N-labelled biomass of microalgae on a commercial scale is presented, and 97.8% of the supplied

nitrogen is assimilated into the biomass [23]. However, in the present paper, 15 N-labeling abundance
of the 15 N-PSTs extract was 82.36%, lower than the abundance of whole cell in existing research [23].
The occurrence of this situation can be explained by the following reasons. Firstly, the time of each
generation culture is not sufficiently long enough. Secondly, the total 15 N abundance in the crude
extract of the toxin cannot fully represent 15 N in the pure toxin, because extract impurities (e.g., protein
and pigment) can cause interference. Thirdly, trace amounts of nitrogen in natural seawater and the
culture solution reagent may affect 15 N-labeled PSTs.

4. Conclusions
     This paper provides the initial evidence that 15 N isotope is feasible to label PSTs in A. minutum
and worthy of being a powerful tool to conduct PST-related researches. In our study, 15 N abundance,
PSTs content and profile were detected and the 15 N atom number introduced into GTX1-4 should be
4–6. However, it is a pity that the precursor and the biosynthetic intermediates of PSTs in A. minutum
were not analyzed. Further study is needed to apply isotope-labeling on both toxin-producing algae
and vector mollusk species so that we can better elucidate the mechanism of PSTs biosynthesis
and metabolism.

5. Materials and Methods

5.1. Chemicals and Analytical Standards
      15 N-NaNO
               3 (δ15 N = 99.14%) was obtained from SRICI (Shanghai Research Institute of Chemical
Industry CO., LTD., Shanghai, China). Bio-gel P-2 (400 mesh), Bio-Rex 70 (400 mesh) were obtained
from BIO-RAD (Hercules, CA, USA). Certified reference materials for PSTs, including gonyautoxin 1/4
(GTX 1/4), gonyautoxin 2/3 (GTX 2/3), were purchased from the National Research Council, Institute
for Marine Bioscience (Halifax, Canada). Analytical grade solvents were used for extraction purposes
while LC grade solvents were used for HPLC-FLD applications.

5.2. Algal Culture
     The PSTs-producing dinoflagellate A. minutum (strain AGY-H46, purchased from Leadingtec,
Shanghai, China) was cultivated in thermo regulated rooms (25 ± 1 ◦ C) with filtered (0.45 µm, Jinjing
Ltd., China) and sterilized (121 ◦ C, 20 min) seawater before enrichment with f/2 medium amendments
(Table 3). The light intensity was set at 3000–4000 lux with a dark:light cycle of 14:10 h. The seawater
was obtained from Donghai Island waters (Zhanjiang, China). Algal cell densities were determined
by optical microscope and cells were collected at particular time for 15 N abundance analysis and
PSTs detection.

                                     Table 3. f/2 medium amendments.

                       Reagent                       Working Solution (mg/L)        Stock Solution (g/L)
             A:                  NaNO3                           75                           75
             B:              NaH2 PO4 ·H2 O                       5                            5
             C:              Na2 SiO3 ·9H2 O                     20                           20
             D:                Na2 EDTA                         4.36                        4.36
             E:               FeCl3 ·6H2 O                      3.16                        3.16
             F:               CuSO4 ·5H2 O                      0.01                        0.01
                              ZnSO4 ·7H2 O                     0.023                        0.023
                              CoCL2 ·6H2 O                     0.012                        0.012
                              MnCL2 ·4H2 O                     0.18                          0.18
                             Na2 MoO4 ·2H2 O                   0.07                          0.07
Toxins 2019, 11, 211                                                                                 9 of 13

                                                   Table 3. Cont.

                          Reagent                        Working Solution (mg/L)   Stock Solution (g/L)
               G:                   Vitamin B1                          0.1                0.01
                                    Vitamin B12                     0.5 × 10−3          0.5 × 10−4
                                     Vitamin H                      0.5 × 10−3          0.5 × 10−4

5.3.   15 N-PSTs    Extraction
      An aliquot (60 mL) of the algal fluid was centrifuged at 6000 r/min under 4 ◦ C for 10 min, and the
supernatant was carefully discarded. The sedimentary cells were resuspended with 0.05 M acetic acid
and then broken using ultrasonic processor in an ice bath for 10 min (power 80%, working 3 s, gap
3 s) until there was no whole cell. The combined liquid was centrifuged at 12000 r/min under 4 ◦ C
for 10 min, then the supernatant was filtered (0.22 µm, Jinjing Ltd., China) and stored under −20 ◦ C
for purification.

5.4.   15 N-PSTs    Separation and Purification
     Separation and purification were carried on by reference to published papers [41,42]. The PSTs
extract was adsorbed to a column of Bio-Gel P-2 equilibrated with water. Furthermore, the column
was first washed with a sufficient volume of water and eluted with 0.1 mM acetic acid at a flow
rate of 0.5 mL/min. After separation, toxin mixture was purified by ion exchange chromatography
using a column of Bio-Rex 70 equilibrated with water and gradient eluted with acetic acid (acetic acid
concentration was as follows: 0, 0.05, 0.055 and 0.060 mM). The fraction was collected every 12 min
and then analyzed by FFA to assure purification efficiency.

5.5.   15 N   Abundance Analysis of 15 N-PSTs Extracts
       15 N
          abundance was analyzed by MAT-271 Gas isotope mass spectrometer (Finnigan, Santa
Clara Valley, CA, USA). Operating conditions were set to high voltage (10 kV), emission current
(0.040 mA), electronic energy (100 eV) and well voltage (134 eV). 15 N-PSTs extraction after freeze-drying
was converted to gas by micro-high-heat combustion method, then entered into gas isotope mass
spectrometer sample introduction system via sample adapter. Mass spectrometer vacuum was less
than 2 × 10−5 Pa. After making the necessary calibration settings for the instrument, the instrument
background measurement was performed. The sample gas was introduced into the sample storage
system at a pressure of 5–10 Pa. The measurement process was automatically performed by computer
instructions, and the signal strength (in mV) of mass number 28, 29, 30 was output as I28 , I29 and I30 .

5.6. PSTs Toxicity Test by the Mouse Bioassay
     The mouse bioassay (MBA) was a referenced method from [43]. 10 healthy male mice were
injected intraperitoneally with 1 mL aliquot of 15 N-PSTs extracts and observed to quantify the toxin
according to the time of death. The toxicity was expressed in mouse units (MU), 1 MU representing
the average toxin amount to kill a mouse weighing 20 g within 15 min.

5.7.   15 N-PSTs    Detection by the Fast Fluorimetric Assay (FFA)
     The fast fluorimetric assay (FFA) was performed by fluorospectrophotometer (HITACHI, Japan)
at a 333 nm excitation wavelength, 10 nm excitation slit, emission wavelength 390 nm and 20 nm
emission slit. The method was from [44] and got some modification in this paper. A portion (0.5 mL)
of each extract or blank solution (0.05 M acetic acid), respectively, was mixed with 2 mL of oxidation
solution (50 mM dipotassium phosphate with 10 mM periodic acid) and incubated for 15 min at
50 ◦ C. After incubation, the reaction mixture was neutralized with 2.5 mL of 1 M acetic acid and
transferred to a cuvette for detection by the fast fluorimetric assay (FFA). Relative fluorescence units
Toxins 2019, 11, 211                                                                                                  10 of 13

(RFU) was recorded and this experiment was conducted to get an overview of the fluorescence of
oxidized samples, but not for accurate quantification.

5.8.   15 N-PSTs   Determination by HPLC-FLD
      HPLC-FLD analysis was carried out using Agilent 1100 (Agilent, Santa Clara, CA, USA) coupled
to PCX5200 (Pickering Laboratories Company) post-column reactor and Agilent G1321A detector
(λex = 330 nm, λem = 390 nm). Chromatographic separation of compounds was achieved on a
reversed-phase C8 column (150 mm × 4.6 mm i.d.; 5 µm, GL Sciences, Tokyo, Japan). The system was
managed by an Agilent Chem. Station 2.1 workstation. The PST extract was subjected to HPLC-FLD
after ultrafiltration (10,000 Da ultrafiltration centrifuge tube, 12,000 r/min for 10 min at 4 ◦ C). Analysis
method was performed according to [45]. The oxidant solution was 50 mM potassium hydrogen
phosphate containing 7 mM periodic acid. The acidifying agent was 0.5 M acetic acid. The flow rates
were 0.8 mL/min for the elution solution and 0.4 mL/min for the oxidant and acidifying agent.

5.9.   15 N-PSTs   Quantification by HPLC-MS
     HPLC-MS analysis was carried out on Agilent 1100 (Agilent, USA) coupled to Waters FIN_C2-XS
QTof mass spectrometer (Waters, Milford, MA, USA) with an electrospray ionization interface. PSTs
were separated on a TSKgel Amide-80 HILIC column (250 mm × 2 mm i.d.; 5 µm, Tosoh Bioscience,
LLC, Montgomeryville, PA, USA). The PSTs extract after purification was subjected to HPLC-MS after
ultrafiltration (10000 Da ultrafiltration centrifuge tube, 12000 r/min for 10 min at 4 ◦ C). A binary mobile
phase included “solvent A” and “solvent B”, in which “solvent A” was water containing 2.0 mM
ammonium formate containing 0.1% (v/v) formic acid and “solvent B” was acetonitrile containing
0.1% (v/v) formic acid. Parameters of mass spectrometer were multiple reaction monitoring (MRM)
mode, positive polarity of ESI, capillary voltage (3.2 kV), ion source temperature (150 ◦ C), desolvation
temperature (400 ◦ C), cone gas flow (50 L/h) and desolvation gas flow (700 L/h).

Author Contributions: Methodology, X.Y.; investigation, M.L., R.Z. and X.H.; writing-original draft preparation,
M.L.; writing-review and editing, W.X.; supervision, L.S., and C.L.; project administration, W.X. and X.Y.
Funding: This research was funded by National Natural Science Foundation of China, grant number 31271938
and 31772089, and Shandong Province Key Research and Development Program, grant number 2017GHY15127.
This work was also financially supported by Major Scientific Research Projects in Universities Cultivation Plan
(Q15137) GDOU2015050202.
Acknowledgments: This research was funded by National Natural Science Foundation of China, grant number
31271938 and 31772089, and Shandong Province Key Research and Development Program, grant number
2017GHY15127. This work was also financially supported by Major Scientific Research Projects in Universities
Cultivation Plan (Q15137) GDOU2015050202. We gratefully acknowledge the help of the other members in our
lab, who have offered us valuable suggestions in the academic studies. Besides, we also show our gratitude to
Shanghai Research Institute of Chemical Industry CO., LTD., which provided technical support for us.
Conflicts of Interest: The authors declare no conflict of interest.

References
1.     Hoagland, P.A.; Anderson, D.M.; Kaoru, Y.; White, A.W. The Economic Effects of Harmful Algal Blooms
       in the United States: Estimates, Assessment Issues, and Information Needs. Estuaries 2002, 25, 819–837.
       [CrossRef]
2.     Gustaafm, H. Ocean climate change, phytoplankton community responses, and harmful algal blooms:
       A formidable predictive challenge. J. Phycol. 2010, 46, 220–235. [CrossRef]
3.     Rolton, A.; Vignier, J.; Soudant, P.; Shumway, S.E.; Bricelj, V.M.; Volety, A.K. Effects of the red tide dinoflagellate,
       Karenia brevis, on early development of the eastern oyster Crassostrea virginica and northern quahog Mercenaria
       mercenaria. Aquat. Toxicol. 2014, 155, 199–206. [CrossRef] [PubMed]
4.     Brooks, B.W.; Lazorchak, J.M.; Howard, M.D.; Johnson, M.V.; Morton, S.L.; Perkins, D.A.; Reavie, E.D.;
       Scott, G.I.; Smith, S.A.; Steevens, J.A. Are harmful algal blooms becoming the greatest inland water quality
       threat to public health and aquatic ecosystems? Environ. Toxicol. Chem. 2016, 35, 6–13. [CrossRef] [PubMed]
Toxins 2019, 11, 211                                                                                             11 of 13

5.    Grattan, L.M.; Holobaugh, S.; Morris, J.G. Harmful algal blooms and public health. Harmful Algae 2016, 57,
      2–8. [CrossRef] [PubMed]
6.    Zhang, Y.; Zhang, S.F.; Lin, L.; Wang, D.Z. Whole Transcriptomic Analysis Provides Insights into Molecular
      Mechanisms for Toxin Biosynthesis in a Toxic Dinoflagellate Alexandrium catenella (ACHK-T). Toxins 2017, 9,
      213. [CrossRef]
7.    Li, A.; Chen, H.; Qiu, J.; Lin, H.; Gu, H. Determination of multiple toxins in whelk and clam samples collected
      from the Chukchi and Bering seas. Toxicon 2016, 109, 84–93. [CrossRef]
8.    Jiang, T.; Xu, Y.; Li, Y.; Jiang, T.; Wu, F.; Zhang, F. Seasonal dynamics of Alexandrium tamarense and occurrence
      of paralytic shellfish poisoning toxins in bivalves in Nanji Islands, East China Sea. Mar. Freshw. Res. 2014, 65,
      350. [CrossRef]
9.    Fabioux, C.; Sulistiyani, Y.; Haberkorn, H.; Hegaret, H.; Amzil, Z.; Soudant, P. Exposure to toxic Alexandrium
      minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas. Harmful
      Algae 2015, 48, 55–62. [CrossRef]
10.   Xie, W.; Liu, X.; Yang, X.; Zhang, C.; Bian, Z. Accumulation and depuration of paralytic shellfish poisoning
      toxins in the oyster Ostrea rivularis Gould—Chitosan facilitates the toxin depuration. Food Control 2013, 30,
      446–452. [CrossRef]
11.   Ding, L.; Qiu, J.; Li, A. Proposed Biotransformation Pathways for New Metabolites of Paralytic Shellfish
      Toxins Based on Field and Experimental Mussel Samples. J. Agric. Food Chem. 2017, 65, 5494–5502. [CrossRef]
      [PubMed]
12.   Mat, A.M.; Klopp, C.; Payton, L.; Jeziorski, C.; Chalopin, M.; Amzil, Z.; Tran, D.; Wikfors, G.H.; Hegaret, H.;
      Soudant, P.; et al. Oyster transcriptome response to Alexandrium exposure is related to saxitoxin load and
      characterized by disrupted digestion, energy balance, and calcium and sodium signaling. Aquat. Toxicol.
      2018, 199, 127–137. [CrossRef] [PubMed]
13.   Han, S.; Yan, S.; Chen, K.; Zhang, Z.; Zed, R.; Zhang, J.; Song, W.; Liu, H. 15 N isotope fractionation in an
      aquatic food chain: Bellamya aeruginosa (Reeve) as an algal control agent. J. Environ. Sci. 2010, 22, 242–247.
      [CrossRef]
14.   Chahrour, O.; Cobice, D.; Malone, J. Stable isotope labelling methods in mass spectrometry-based quantitative
      proteomics. J. Pharm. Biomed. Anal. 2015, 113, 2–20. [CrossRef] [PubMed]
15.   Brandsma, J.; Bailey, A.P.; Koster, G.; Gould, A.P.; Postle, A.D. Stable isotope analysis of dynamic lipidomics.
      Biochim. Biophys. Acta 2017, 1862, 792–796. [CrossRef]
16.   Schneider, L.; Maher, W.A.; Potts, J.; Taylor, A.M.; Batley, G.E.; Krikowa, F.; Adamack, A.; Chariton, A.A.;
      Gruber, B. Trophic transfer of metals in a seagrass food web: Bioaccumulation of essential and non-essential
      metals. Mar. Pollut. Bull. 2018, 131, 468. [CrossRef] [PubMed]
17.   He, Y.-L.; Luo, Y.-B.; Chen, H.; Hou, H.-W.; Hu, Q.-Y. Research Progress in Analysis of Small
      Molecule Metabolites in Bio-matrices by Stable Isotope Coded Derivatization Combining with Liquid
      Chromatography–tandem Mass Spectrometry. Chin. J. Anal. Chem. 2017, 45, 1066–1077. [CrossRef]
18.   Thomas, M.; Huck, N.; Hoehenwarter, W.; Conrath, U.; Beckers, G.J.M. Combining Metabolic 15 N Labeling with
      Improved Tandem MOAC for Enhance; Springer: New York, NY, USA, 2015; pp. 81–96.
19.   Lehmann, W.D. A timeline of stable isotopes and mass spectrometry in the life sciences. Mass Spectrom. Rev.
      2016, 36. [CrossRef]
20.   Bequette, B.J.; Backwell, F.R.C.; Calder, A.G.; Metcalf, J.A.; Beever, D.E.; Macrae, J.C.; Lobley, G.E. Application
      of a U-13 C-Labeled Amino Acid Tracer in Lactating Dairy Goats for Simultaneous Measurements of the Flux
      of Amino Acids in Plasma and the Partition of Amino Acids to the Mammary Gland. J. Dairy Sci. 1997, 80,
      2842–2853. [CrossRef]
21.   Cox, J.; Kyle, D.; Radmer, R.; Delente, J. Stable-isotope-labeled biochemicals from microalgae. Trends
      Biotechnol. 1988, 6, 279–282. [CrossRef]
22.   Fernández, F.G.A.; Alias, C.B.; Pérez, J.A.S.; Sevilla, J.M.F.; González, M.J.I.; Grima, E.M. Production of 13 C
      polyunsaturated fatty acids from the microalga Phaeodactylum tricornutum. J. Appl. Phycol. 2003, 15, 229–237.
      [CrossRef]
23.   Acien Fernandez, F.G.; Fernandez Sevilla, J.M.; Egorova-Zachernyuk, T.A.; Molina Grima, E. Cost-effective
      production of 13 C, 15 N stable isotope-labelled biomass from phototrophic microalgae for various
      biotechnological applications. Biomol. Eng. 2005, 22, 193–200. [CrossRef] [PubMed]
Toxins 2019, 11, 211                                                                                             12 of 13

24.   Carcelén, J.N.; Marchante-Gayón, J.M.; González, P.R.; Valledor, L.; Cañal, M.J.; Alonso, J.I.G. A cost-effective
      approach to produce 15 N-labelled amino acids employing Chlamydomonas reinhardtii CC503. Microb. Cell
      Factories 2017, 16, 146. [CrossRef] [PubMed]
25.   Wiese, M.; D’Agostino, P.M.; Mihali, T.K.; Moffitt, M.C.; Neilan, B.A. Neurotoxic alkaloids: Saxitoxin and its
      analogs. Mar. Drugs 2010, 8, 2185–2211. [CrossRef] [PubMed]
26.   Hwang, D.F.; Lu, Y.H. Influence of environmental and nutritional factors on growth, toxicity, and toxin
      profile of dinoflagellate Alexandrium minutum. Toxicon 2000, 38, 1491–1503. [CrossRef]
27.   Hui, G.; Jianting, Y.; Zhongmin, S.; Delin, D. Effects of salinity and nutrients on the growth and chlorophyll
      fluorescence of Caulerpa lentillifera. Chin. J. Oceanol. Limnol. 2015, 33, 410–418. [CrossRef]
28.   Boyer, G.L.; Sullivan, J.J.; Andersen, R.J.; Harrison, P.J.; Taylor, F.J.R. Effects of nutrient limitation on toxin
      production and composition in the marine dinoflagellate Protogonyaulax tamarensis. Mar. Boil. 1987, 96,
      123–128. [CrossRef]
29.   Hamasaki, K.; Horie, M.; Tokimitsu, S.; Toda, T.; Taguchi, S. Variability in Toxicity of the Dinoflagellate
      Alexandrium Tamarense Isolated from Hiroshima Bay, Western Japan, as a Reflection of Changing
      Environmental Conditions. J. Plankton Res. 2001, 23, 271–278. [CrossRef]
30.   Wang, D.-Z.; Hsieh, D.P.H. Growth and toxin production in batch cultures of a marine dinoflagellate
      Alexandrium tamarense HK9301 isolated from the South China Sea. Harmful Algae 2005, 4, 401–410. [CrossRef]
31.   Anderson, D.M.; Kulis, D.M.; Sullivan, J.J.; Hall, S.; Lee, C. Dynamics and physiology of saxitoxin production
      by the dinoflagellates Alexandrium spp. Mar. Boil. 1990, 104, 511–524. [CrossRef]
32.   Dell’Aversano, C.; Hess, P.; Quilliam, M.A. Hydrophilic interaction liquid chromatography–mass
      spectrometry for the analysis of paralytic shellfish poisoning (PSP) toxins. J. Chromatogr. A 2005, 1081,
      190–201. [CrossRef] [PubMed]
33.   Guéguen, M.; Baron, R.; Bardouil, M.; Truquet, P.; Haberkorn, H.; Lassus, P.; Barillé, L.; Amzil, Z. Modelling
      of paralytic shellfish toxin biotransformations in the course of Crassostrea gigas detoxification kinetics.
      Ecol. Model. 2011, 222, 3394–3402. [CrossRef]
34.   Brandsma, J.; Postle, A.D. Analysis of the regulation of surfactant phosphatidylcholine metabolism using
      stable isotopes. Ann. Anat. Anat. Anz. 2017, 211, 176–183. [CrossRef] [PubMed]
35.   Cho, Y.; Tsuchiya, S.; Omura, T.; Koike, K.; Oikawa, H.; Konoki, K.; Oshima, Y.; Yotsu-Yamashita, M.
      Metabolomic study of saxitoxin analogues and biosynthetic intermediates in dinoflagellates using
      (15)N-labelled sodium nitrate as a nitrogen source. Sci. Rep. 2019, 9, 3460. [CrossRef] [PubMed]
36.   Roelke, D.L.; Eldridge, P.M.; Cifuentes, L.A. A Model of Phytoplankton Competition for Limiting and
      Nonlimiting Nutrients: Implications for Development of Estuarine and Nearshore Management Schemes.
      Estuaries 1999, 22, 92–104. [CrossRef]
37.   Hii, K.S.; Lim, P.T.; Kon, N.F.; Takata, Y.; Usup, G.; Leaw, C.P. Physiological and transcriptional responses to
      inorganic nutrition in a tropical Pacific strain of Alexandrium minutum: Implications for the saxitoxin genes
      and toxin production. Harmful Algae 2016, 56, 9–21. [CrossRef] [PubMed]
38.   Lim, P.T.; Leaw, C.P.; Kobiyama, A.; Ogata, T. Growth and toxin production of tropical Alexandrium minutum
      Halim (Dinophyceae) under various nitrogen to phosphorus ratios. J. Appl. Phycol. 2010, 22, 203–210.
      [CrossRef]
39.   Sauer, M.L.A.; Xu, B.; Sutton, F. Metabolic labeling with stable isotope nitrogen ( 15 N) to follow amino
      acid and protein turnover of three plastid proteins in Chlamydomonas reinhardtii. Proteome Sci. 2014, 12, 1–9.
      [CrossRef]
40.   Chou, H.N.; Chen, Y.M.; Chen, C.Y. Variety of PSP toxins in four culture strains of Alexandrium minutum
      collected from southern Taiwan. Toxicon 2004, 43, 337–340. [CrossRef]
41.   Miao, Y.P.; Zhou, H.N.; Wen, R. Isolation and purification of gonyautoxins from Alexandrium mimutum Halim.
      Acta Pharm. Sin. 2004, 39, 52–55. [CrossRef]
42.   Laycock, M.V.; Thibault, P.; Ayer, S.W.; Walter, J.A. Isolation and purification procedures for the preparation
      of paralytic shellfish poisoning toxin standards. Neurogastroenterol. Motil. 2010, 2, 175–183. [CrossRef]
43.   AOAC. AOAC Official Method 959.08: Paralytic Shellfish Poison, Biological Method; AOAC: Arlington, VA,
      USA, 2000.
Toxins 2019, 11, 211                                                                                       13 of 13

44.   Gerdts, G.; Hummert, C.; Donner, G.; Luckas, B.; Schütt, C. A fast fluorimetric assay (FFA) for the detection
      of saxitoxin in natural phytoplankton samples. Mar. Ecol. Prog. 2002, 230, 29–34. [CrossRef]
45.   Van De Riet, J.; Gibbs, R.S.; Muggah, P.M.; Rourke, W.A.; Macneil, J.D.; Quilliam, M.A. Liquid chromatography
      post-column oxidation (PCOX) method for the determination of paralytic shellfish toxins in mussels, clams,
      oysters, and scallops: Collaborative study. J. AOAC Int. 2011, 94, 1154–1176. [PubMed]

                        © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
                        article distributed under the terms and conditions of the Creative Commons Attribution
                        (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
You can also read